The engagement of the T cell receptor (TCR) on T lymphocytes initiates signaling pathways that regulate cell proliferation, activation, and survival through the activation of transcription factors that regulate programs of gene expression. TCR signaling is initiated when an antigen presenting cell (APC) presents antigen, in the appropriate molecular context, to a T cell. After APC:T cell conjugation, the immunological synapse forms in the region of cell:cell contact. Signaling from the synapse to transcription factors requires the recruitment of signaling intermediates to the immunological synapse in a process that is incompletely understood. CARD11 is a multi-domain adapter protein that coordinates the signal-induced association of a set of proteins that are required for TCR-mediated activation of NF-kappaB. In order to expand our understanding of how CARD11 relays signals from the TCR to NF-kappaB, we have developed a novel expression cloning strategy for the identification of modulators of CARD11 signaling activity. Using this strategy, we isolated the kinesin-like motor protein GAKIN as a negative regulator of CARD11. In our preliminary studies, we have determined that GAKIN overexpression inhibits CARD11 activity and TCR signaling while the reduction in GAKIN expression results in enhanced CARD11 and TCR signaling. GAKIN and CARD11 associate at endogenous levels in T cells in a signal-inducible manner. Imaging studies suggest that the cellular localization of GAKIN changes during TCR signaling, which may depend on GAKIN's ability as a motor protein to move cargo along microtubules. In this application, we propose to test our overall hypothesis that GAKIN is a critical negative regulator of TCR signaling that regulates the scaffolding function and cellular localization of CARD11. Using biochemical approaches, we will investigate which domains of GAKIN are required for its association with CARD11 and for its ability to negatively regulate TCR signaling. We will also determine whether GAKIN modulates the association of signaling cofactors with CARD11. In imaging experiments, we will characterize how the localization of GAKIN is determined and whether GAKIN regulates the recruitment of CARD11 and signaling factors to the immunological synapse. GAKIN associates with CARD11 in a region in which oncogenic mutations have been found in Diffuse Large B cell Lymphoma (DLBCL). We will investigate whether these mutations affect GAKIN-mediated regulation of CARD11 activity and determine whether GAKIN can inhibit the dysregulated growth in DLBCL. Our results should add to the understanding of how the molecular machinery of immune cells can recognize and interpret environmental cues, including pathogenic and nonpathogenic stimuli, and respond appropriately. Since this machinery is impaired in aged T cells, dysregulated in immunodeficiencies, and is abnormally hyperactive in autoimmune disease and in several types of cancer, our results may illuminate molecular targets for the development of new therapies designed to treat multiple diseases of the immune system. PUBLIC HEALTH RELEVANCE: The normal function of the immune system depends on the ability of white blood cells, or lymphocytes, to detect foreign pathogens and respond appropriately so that a pathogen is eliminated without damage to the host. This proposal is designed to expand understanding of the molecular machinery that is used by T lymphocytes in this process. Since this machinery is impaired in aged T cells, dysregulated in immunodeficiencies, and is abnormally hyperactive in autoimmune disease and in several types of cancer, our results may illuminate molecular targets for the development of new therapies designed to treat multiple diseases of the immune system.